丝胶可激活2型糖尿病大鼠蛋白激酶B信号转导通路减少海马神经元的凋亡

1、NEURAL REGENERATION RESEARCH Volume 7, Issue 3, January 2012Cite this article as: Neural Regen Res. 2012;7(3:197-201.197Zhihong Chen , Doctor, Professor, Department of Human Anatomy, Chengde Medical College, Chengde 067000, Hebei Province, ChinaCorresponding author: Jing-feng Xue, Master, Professor,

2、 Department of HumanAnatomy, Chengde Medical College, Chengde 067000, Hebei Province, China xjfReceived: 2011-09-19 Accepted: 2011-11-15 (N20110723001/YJChen ZH, He YQ, Song CJ, Dong ZJ, Su ZJ, Xue JF. Sericin can reduce hippocampal neuronal apoptosis by activating the Akt signal transduction pathwa

3、y in a rat model of diabetes mellitus. Neural Regen Res.2012;7(3:197-201.doi:10.3969/j.issn.1673-5374.2012.03.007Sericin can reduce hippocampal neuronal apoptosis by activating the Akt signal transduction pathway in a rat model of diabetes mellitus*Zhihong Chen 1, Yaqiang He 1, Chen

4、gjun Song 1, Zhijun Dong 2, Zhejun Su 2, Jingfeng Xue 11Department of Human Anatomy, Chengde Medical College, Chengde 067000, Hebei Province, China 2Affiliated Hospital of Chengde Medical College, Chengde 067000, Hebei Province, ChinaAbstractIn the present study, a rat model of type 2 diabetes melli

5、tus was established by continuousperitoneal injection of streptozotocin. Following intragastric perfusion of sericin for 35 days, blood glucose levels significantly reduced, neuronal apoptosis in the hippocampal CA1 region decreased, hippocampal phosphorylated Akt and nuclear factor kappa B expressi

6、on were enhanced, butBcl-xL/Bcl-2 associated death promoter expression decreased. Results demonstrated that sericin can reduce hippocampal neuronal apoptosis in a rat model of diabetes mellitus by regulating abnormal changes in the Akt signal transduction pathway.Key Words: sericin; type 2 diabetes

7、mellitus; hippocampus; apoptosis; Akt signal transduction pathway; neural regenerationINTRODUCTIONOne floss from the cocoon shell of the Bombyx mori silkworm, a member of the Bombycidae family 1, is composed of two parallel monofilament fibers. Eachmonofilament has a silk protein center that is surr

8、ounded by sericin. To date, most studies have focused on the application of sericin protein in cosmetology, skin care, nutrition, as an anti-oxidant and as a potential anticancer therapy 2-3. Our previous studies have demonstrated that sericin can effectively protect islet cells, the testis and the

9、kidney from injury in diabetic rats 4-6. The protective effects of sericin on nervous system lesions in diabetes mellitus remain poorly understood, however, sericin has exhibitedneuroprotective effects in a rat model of diabetes mellitus by inhibiting cortical and hippocampal heme oxygenase1 express

10、ion 7. The hippocampus is sensitive to chronic hyperglycemia in diabetes mellitus, which manifests as a reduction in hippocampal neuron number, abnormal morphology, learning and cognitive dysfunction, delayed responses to surroundings, and cognitive impairment characterized by acquired cognitive and

11、 behavioral deficiency 8-9. However, in rats with diabetes mellitus and cognitive impairment, the number ofapoptotic neurons in the hippocampal CA1 region was significantly increased, indicating neuronal apoptosis participates inhippocampal injury-induced cognitiveimpairment following diabetes melli

12、tus 10. The Akt signal transduction pathway is composed of Akt, nuclear factor kappa B (NF-B and the Bcl-xL/Bcl-2 associated death promoter (Bad, and is involved in cell proliferation, apoptosis, differentiation and metabolism 11-12. Akt is extensivelyexpressed in tissues, and phosphorylated Akt (p-

13、Akt plays a role in cell metabolism, survival, proliferation, apoptosis, cell cycle regulation, and angiopoiesis 13-15. p-Akt can phosphorylate the inhibitory protein of NF-B (IB by activating the inhibitoryprotein of NF-B kinase to dissociate NF-B from NF-B -IB. This restores NF -B ac tivity, and i

14、nhibits nerve cell apoptosis 16-17. In addition, p-Akt phosphorylates Bad.Activated Bad can bind to the chaperonin protein 14-3-3 to block dimer formation of Bad and Bcl-2/or Bcl-xL 18-19. Therefore, the activated Akt signal transduction pathway can relieve hippocampal neuronal injury 20. The presen

15、t study investigated the effects of sericin on the hippocampal Akt signaltransduction pathway in a rat model of type 2 diabetes mellitus to confirm the protective effects of sericin on hippocampal injury in type 2 diabetes mellitus.RESULTSQuantitative analysis of experimental animalsA total of 30 ad

16、ult Sprague-Dawley rats 198used. Ten were selected for the control group, and the remaining 20 were used to establish a model of type 2 diabetes mellitus, and subdivided into model (no treatment and sericin (sericin perfusion groups. All 30 rats were included in the final analys

17、is. Sericin reduced blood glucose levels in rats The blood glucose levels in model rats were significantly reduced following sericin treatment for 35 days (P < 0.01; Table 1. Sericin inhibited neuronal apoptosis in the hippocampal CA1 region of rats Terminal deoxynucleotidyl transferase-mediated

18、dUTP nick end labeling (TUNEL detection showed neuronal apoptosis in the hippocampal CA1 region of each group. The number of apoptotic neurons was significantly elevated in model rats when compared with the control group (P < 0.01. Following sericin treatment for 35 days, the number of apoptotic

19、neurons significantly decreased (P < 0.05; Table 1, Figure 1. Sericin promoted p-Akt, NF-B protein and mRNA expression, but inhibited Bad protein and mRNA expression in the hippocampus of rats Protein and mRNA levels of p-Akt, NF-B and Bad in thehippocampus were detected using western blotting an

20、dreverse transcription (RT-PCR. p-Akt and NF-B protein and mRNA expression was significantly reduced in the hippocampus of rats with diabetes mellitus (P < 0.01, while Bad protein and mRNA levels were significantly elevated (P < 0.01. Following sericin treatment for 35 days, p-Akt and NF-B pro

21、tein and mRNA expression was significantly increased (P < 0.01, but Bad protein and mRNA levels were significantly decreased in the hippocampus (P < 0.01; Figure 2, Table 2. Figure 1 Neuronal apoptosis in the hippocampus (TUNEL, × 200.Apoptotic neurons had brown/yellow stained nuclei. Com

22、pared with the control group (A, the number of apoptotic neurons in the hippocampal CA1 region was significantly increased in the model group (B, but the sericin group (C exhibited significantly less apoptotic neurons in the hippocampal CA1 region. TUNEL: Terminal deoxynucleotidyl transferase-mediat

23、ed dUTP nick end labeling. A B C Protein and mRNA levels of phosphorylated Akt (p-Akt; A, nuclear factor kappa B (NF-B; B and Bcl-xL/Bcl-2associated death promoter (Bad; C expression in the hippocampus using western blotting and reverse transcription-PCR. p-Akt B protein and mRNA expression was sign

24、ificantly increased following sericin treatment for 35 days. Bad protein and mRNA expression was significantly decreased following sericin treatment for 35 days. 1: Control group; 2: model group; 3: sericin group; B 12 3 12 3 12 3 Marker 1 2 3 Marker 1 2 3 Marker 1 2 3 p-Akt 1 500 bp 1 000 bp 850 bp

25、750 bp 650 bp 550 bp 450 bp350 bp 250 bp 150 bp 1 500 bp 1 000 bp 850 bp 750 bp 650 bp 550 bp 450 bp 350 bp 250 bp 150 bp DISCUSSIONIn the present study, the hippocampal Akt signal transduction pathway exhibited abnormal changes in rats with diabetes mellitus, as manifested by decreased p-Akt and NF

26、-B expression and elevated Bad expression. Hyperglycemia in diabetes mellitus can inhibit Akt activation or accelerate Akt deactivation, and reduce Akt phosphorylation by activating the c-Jun N-terminal kinase pathway21-22. Moreover, hyperglycemia in diabetes mellitus can induce hippocampal blood ca

27、pillary injury, leading to regional ischemia and elevated Bad expression levels23-24. In the present study, p-Akt and NF-B expre ssion was significantly elevated, but Bad expression was significantly reduced in the sericin group when compared with the model group. Interestingly, increased p-Akt expr

28、ession has been shown to induce an anti-apoptotic effect, and phosphorylate and activateNF-B16-17 and Bad18-19.Therefore, results from the present study indicated that sericin can reduce hippocampal neuronal apoptosis and protect the hippocampus against injury by regulating the Akt signal transducti

29、on pathway in rats with diabetes mellitus. Moreover, sericin is a natural protein and will reduce the possibility of toxic side effects that can occur from chemically synthesized drugs. However, the mechanism underlying how sericin regulates the Akt signal transduction pathway needs further investig

30、ation. MATERIALS AND METHODSDesignA randomized, controlled, animal study.Time and settingThe experiment was performed at the Institute of Basic Medicine, Chengde Medical College, China, from June 2009 to October 2010.MaterialsAnimalsA total of 30 healthy, male, Sprague-Dawley rats, aged 3 months, we

31、ighing 200-250 g, were provided by the Laboratory Animal Center of Hebei Medical University (No. 712024. They were housed at 20 ± 2°C in 40-70% humidity. The experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formu

32、lated by the Ministry of Science and Technology of China25.DrugsSericin was made from the colored silkworm cocoon (Sericultural Research Institute, Chengde Medical College and was prepared by soaking, decocting, filtrating and condensing, according to a previously described method7.MethodsEstablishm

33、ent of type 2 diabetes mellitus and sericin administrationThe rat model of diabetes mellitus was established by intraperitoneal injection of 2% (w/v streptozotocin(25 mg/kg; Sigma, St. Louis, MO, USA for 3 consecutive days. A successful model was demonstrated when rats had blood glucose levels 16.7

34、m M26. Sericin group rats were intragastrically perfused with sericin, 2.4 g/kg per day immediately following model establishment27 for 35 consecutive days.Blood glucose detectionBlood glucose levels in the model group were detected following model establishment, and were detected 35 days following

35、sericin administration in the sericin group. The rats were fasted for 12 hours or more, and anesthetized by intraperitoneal injection of 4% (w/v chloral hydrate. Blood (about 3 mL was collected from the medial angle of the eye, and centrifuged at3 000 r/min for 20 minutes. The supernatant was collec

36、ted, and blood glucose levels was detected using the glucose oxidase method, glucose detection kit (Baoding Great Wall Clinical Reagents Co., Ltd., Baoding, Hebei Province, China; No. 20071030 and Boehringer Mannheim/Hitachi 717 automatic clinical biochemical analyzer (Hitachi, Tokyo, Japan. Prepara

37、tion of brain tissue samplesThe brain was harvested after rats were anesthetized and sacrificed. The hippocampus from one hemisphere was rapidly isolated on ice28 and stored in liquid nitrogen, and the hippocampus from the remaining hemisphere was fixed in Bouins solution (75 mL saturated picric aci

38、d199water solution, 25 mL formalin (40% and 5 mL glacial acetic acid.TUNEL detection for neuronal apoptosis in the hippocampal CA1 regionBrain tissues fixed in Bouins solution were paraffin embedded and sectioned (5 m thick. Hippocampal neuronal apoptosis was detected using the apoptosis detection k

39、it (Beijing Zhongshan Goldbridge, Beijing, China29. TUNEL staining results were quantitatively analyzed using the MiVnt image analysis system (Shandong Echuang Electronics, Jinan, Shandong, China. Briefly, three fields of view were selected from the hippocampal CA1 region under a x200 magnification

40、light microscope (Olympus, Tokyo, Japan, and a ratio of positive staining to the total area analyzed was regarded as the apoptotic index of neurons in the hippocampalCA1 region. Five sections were selected from each rat, and 10 rats from each group were used.Western blotting for hippocampal p-Akt, N

41、F-B and Bad protein expressionThe hippocampus (100 mg stored in liquid nitrogen was placed in 1 mL pre-cooled phosphate buffered saline (0.01 mM, pH 7.4, homogenized on ice, centrifuged, and the supernatant was discarded. The sediments were treated with 400 µL of RIPA buffer (RIPA: PMSF = 100:

42、1, left on ice for 40 minutes, and centrifuged at 12 000 r/min at 4°C for 15 minutes. The supernatant was retained. The concentration of protein was quantified using the BCA protein kit (Beijing Taigemei Science and Technology, Beijing, China30. Samples were electrophoresed on a 15% (w/v SDS-po

43、lyacrylamide gel and transferred onto nitrocellulose membrane. Membranes were incubated with rabbit anti-p-Akt polyclonal antibody (1: 100; Beijing Biosynthesis Biotechnology, Beijing, China, mouse anti-NF-B(1: 100, rabbit anti-Bad (1: 200 polyclonal antibody (Beijing Biosynthesis Biotechnology and

44、mouse anti-rat -actin monoclonal antibody (1: 1 000; Santa Cruz Biotechnology, Santa Cruz, CA, USA overnight at 4°C, followed by goat anti-rabbit or mouse IgG (1: 5 000; KPL, Gaithersburg, Maryland, USA at room temperature for 1 hour. Development was performed using Super ECL Plus enhanced chem

45、iluminescence (Beijing Taigemei Science and Technology, Beijing, China. The film was scanned using an EPSON scanner (Epson, Beijing, China, and developed bands were analyzed using QuantityOne-4.6.2 software (Bio-Rad, Hercules, CA, USA. Protein expression levels were determined as the absorbance rati

46、o of the target band to -actin.RT-PCR detection for hippocampal p-Akt, NF-B and Bad mRNA expressionThe hippocampus stored in liquid nitrogen was collected and total RNA was extracted according to the Trizol RNA extraction kit (Invitrogen, Carlsbad, CA, USA. RNA was reverse transcribed into cDNA. PCR

47、 primers were synthesized by Sangon, Shanghai, China.PCR primer sequence and amplification conditions: TA: Annealing temperature; F: forward; R: reverse.PCR conditions: 94°C for 2 minutes; 94°C for 30 seconds; 50-65°C for 30 seconds; 72°C for 1 minute, circulated from the second

48、step. Amplified products, 5 L and DNA Ladder, 5 L (Beijing Taigemei Science and Technology were used for 2% (w/v agarose gel electrophoresis at90 V for 40 minutes. Images were photographed using the ZF UV transmittance analyzer (Shanghai Jiapeng, Shanghai, China and quantitatively analyzed using Qua

49、ntity One-4.6.2 software. Target gene mRNA expression levels were represented by the absorbance ratio of the target band to -actin31.Statistical analysisData were expressed as the mean ± SD and analyzed using SPSS 11.5 software (SPSS, Chicago, IL, USA. Intergroup differences were compared using

50、 one-way analysis of variance, and paired comparison was performed using the q-test. A value of P < 0.05 was considered statistically significant.Author contributions: Zhihong Chen was in charge of funds, conceived and designed the study, analyzed data and wrote the manuscript. Yaqiang He analyze

51、d the data. Chengjun Song fed the animals and provided experimental data. Zhijun Dong and Zhejun Su conducted the statistical analysis. Jingfeng Xue conceived and designed the study, revised the manuscript and guided the study.Conflicts of interest: None declared.Funding: This study was supported by

52、 a grant from the Education Department of Hebei Province (Mechanism ofGH/IGF-1 and protective effects of sericin on gonadal axis lesions in diabetes mellitus, No. 2006301; a grant from Science and Technology Department of Hebei Province (Protective effects of sericin on testicular dysfunction in dia

53、betes mellitus, No. 08276101D-19.Ethical approval: This study received permission from the Animal Ethics Committee of Hebei Province, China. Acknowledgments:We thank the staff from the Institute of Basic Medicine, Chengde Medical University, China, for their help.200REFERENCES1 Li SZ. Compendium of

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